Analysis of T-2 and HT-2 toxin in soybean and soy meal using immunoaffinity clean-up and high performance liquid chromatography

BARROS, G.; GARCÍA, D.; OVIEDO, M.S.; RAMIREZ, M.L.; TORRES, A.; CHULZE, S.

Mérida

Congreso; IV Congreso Latinoamericano de Micotoxicología; 2010

Sociedad Latinoamericana de Micotoxicología

Aim: to evaluate an HPLC analysis coupled with immunoaffinity column clean-up previously described by Visconti et al. (2005) for simultaneous determination of T-2 and HT-2 in cereal grains to be applied in soybean and soy meal samples. Materials and Methods: Fifty grams of sample were extracted with 2 g NaCl and 100 mL of methanol:water (90:10, v v-1) by shaking in a orbital shaker at high speed for 30 min. The extract was filtered through filter paper Whatman Nº 4. Ten milliliters of filtrate were collected and mixed with 40 mL of sodium chloride solution (2%, w v-1). The diluted extract was left on the bench for 5 min. to allow precipitation to occur and then was filtered through a glass microfiber filter. Ten milliliters of filtrate (equivalent to 1.0 g of sample) were passed through the T-2 immunoaffinity column at a flow rate about one drop per second. The column was washed with ten milliliters of deionised water and the mycotoxins eluted from the column using 1.5 mL of methanol. The eluted extract was collected in a 4-mL screw-cap amber vial and evaporated under a stream of nitrogen at 50ºC in a heating block. The dried residue was derivatized with 50 µL of DMAP solution followed by 50 µL of 1-AN reagent as previously described for T-2 and HT-2 toxins by Visconti et al. (2005). Recovery experiments were performed in triplicate by spiking blank soybean and soy meal samples with T-2 and HT-2 toxins at levels of 500, 250 and 125 µg kg-1 by diluting aliquots of the stock solutions with the appropriate volumes of acetonitrile. Spiked samples were left overnight at room temperature to allow solvent evaporation prior extraction. Results and discussion: Characteristics of this in-house method such as accuracy, precision and detection and quantification limits were defined by means of recovery test with spiked soybean and soy meal samples. Mean recoveries for T-2 within the spiking range 125-500 µg/kg, were 90.9 and 81.3% for soybean and soy meal, respectively with a within-laboratory relative standard deviation < 10%. Analysis of samples spiked with HT-2 in the same range gave a mean recovery of 70.2 and 77.5% for soybean and soy meal, respectively, with relative standard deviations < 12%. The limit of detection (LOD) for the method was 25 µg/kg for T-2 and HT-2, based on a signal-to-noise ratio 3:1 and the limit of quantification (LOQ) was established as three times the detection limit. In our study, the analysis of twenty soybean and soy meal samples obtained from an agricultural company located in Córdoba Province, Argentina, revealed that only one soybean sample showed T-2 contamination at level of 279 µg kg-1 but HT-2 contamination was not observed. Conclusion: The proposed HPLC method uses commercially available products, is simple to perform and shows good laboratory performance for determination of T-2 and HT-2. The method provides an alternative to GC as well as HPLC-MS/MS methods for the analysis of samples in those laboratories without mass spectrometry equipment.